Early steps in human immunodeficiency virus (HIV-1) replication involve delivery of the viral core into the cytoplasm of the host cell. When the viral core has reached the cytoplasm a process known as uncoating takes place. Uncoating is the process by which monomeric capsid sheds from the retroviral core. Simultaneously, the viral RNA genome is converted into viral DNA (vDNA), which is subsequently translocated to the nucleus and integrated into the cellular DNA, allowing expression and production of new viral particles. Although the relationship between capsid shedding and synthesis of viral DNA (vDNA) is not understood, the stability of the viral core in the cytoplasm seems to be important for productive infection. Changes in stability of the retroviral core seem to affect vDNA synthesis: 1) mutations in the capsid protein of HIV-1 that diminish (or increase) the stability of the HIV-1 core interfere with vDNA synthesis; 2) TRIM51 proteins block HIV-1 replication by altering the stability of the retroviral core and disrupt vDNA synthesis; 3) HIV-1 cores isolated from particles produced in the absence of accessory proteins such as vif, vpr or nef exhibit low stability and are poorly infectious due to a defect in vDNA synthesis; 4) the use of proteasome inhibitors during HIV-1 infection increases the stability of the core, and at the same time augments the synthesis of vDNA; 5) Isolated HIV-1 cores require cellular extracts in order to undergo vDNA synthesis, indicating the requirement of host factors; and 6) isolated retroviral cores last longer in cytosolic extracts than in isotonic buffer, suggesting the existence of core-stabilizing factors that are essential for vDNA synthesis. Altogether, this evidence indicates that viral core stability and successful vDNA synthesis involve a delicate balance of tightly interwoven events. In this proposal we will test the hypothesis that optimal stability of the retroviral core and cellular factors modulate the rate and extent of uncoating in the cytoplasm, and that this rate and extent of uncoating is required for the occurrence of complete viral DNA synthesis. We will test this hypothesis by performing the following experiments: 1) we will study the effect of factors that destabilize the retroviral core on viral DNA synthesis, 2) we will study the contribution of the proteasome to retroviral uncoating, and 3) we will study the modulation of vDNA synthesis by the interaction of reverse transcriptase with the retroviral ribonucleoprotein complex. The results from these experiments will generate basic knowledge in the uncoating of HIV-1. Uncoating is a poorly explored step on HIV-1 therapeutics; however, we have learned from restriction factors, such as TRIM5, that uncoating is potentially a very effective target for HIV-1 therapeutics.